Team:HBUT-China/Notebook
Notebook
Molecular experiment log
Experimental Objectives:Construction of recombinant plasmids containing the pncrA promoter and the NcrB protein sequence And try to construct a recombinant plasmid containing the complete component.
Figure 1 Plasmid map of bacteria
Figure 2 Bacteria of biological bricks
7.21-7.29
Topic: PCR amplification amplification pncrA and repressor protein NcrB and agar making
Materials:
2 × Buffer 100 μl
DD H2O 92 μl
pncrA forward primer 1 μl
pncrA reverse primer 1 μl
NcrB forward primer 1 μl
NcrB reverse primer 1 μl
The plasmid 4 μl
Agarose 0.3g
1 × TAE 30ml
1/10000 Bio Teke Dye 3 μl
2000bp maker4μl
10 × Buffer 8 μl
Procedure:
1. 5 μl of Buffer, 23 μl of DD H2O, 1 μl of plasmid, and 0.5 μl of forward primer and 0.5 μl reverse primer were mixed into the tube for a total of 50 μl. The mixture was divided and divided into two tubes and placed in a PCR instrument. PCR final system is the 25μl .There are 8 in total, of which 1, 2, 3, 4 marked N; 5,6,7,8 marked P.
PCR parameters set: denaturation 94 ° C, refolding 60 ° C, extended 72 ° C, 30 cycles.2. Take the 0.3 g agarose microwave oven and heat it in 30 ml of 1 × TAE (prepare 1% agarose gel).
3. Add 3μl of dye to the hot mix and Pour into the mold .If bubbles are generated, removeing them with plastic pipettes.
4. Let the gel solidify for 30 min.
5. After the gelation, suit remove the comb. Place it in the gel tank.
6. At the end of the PCR, take out 8 tubes and 8 μl were mixed with 1 μl of 10 × Buffer and added to each well. At the end of PCR, 8 tubes were removed and then 8 μl were mixed with 1 μl of 10 × Buffer and added to each well. When the point of time, the tip of the gun is perpendicular to the filling hole so that the corner just under the opening in the sample hole. Add the sample and the mark maker slowly to the sample hole
7. Run the gel electrophoresis at 114V for 30 minutes.
8. Press the glue
Results and analysis:
Fig 3 .PCR products of pncrA and NcrB in plasmid
Note: M: DL2000 DNA marker
The promoter (pncrA) and repressor (NcrB) were successfully amplified.
1. The first system settings: 50μl system, the primer plus 1μl
Results: There are many non-specific amplification
Analysis: Because the primer concentration is too much, there are many non-specific amplifications
2. The second system settings: 25μl system, the primer plus 0.5μl
Results: As shown in the figure, the nonspecific bands are less, the target bands had no obvious tailing and are clearly visible.
Analysis: The product was successfully amplified and the next step was purified
Topic:DNA recovery
Materials:
Agarose 0.6g
1×TAE 60ml
1/10000 Bio Teke Dye 6 μl
2000bp maker 8μl
10×Buffer 8 μl
Amplified fragment promoter (pncrA)
Amplified fragment repressor protein (NcrB)
DNA purification recovery kit (TIANGEN company)
Procedure:
Use DNA purification recovery kit and then run the glue to see the recovery effect
Results and analysis:
Fig4. Pecturing of pncrA and NcrB
Fig5. Recovery of pncrA and NcrB
Glue recycling are successful and no other specific bands.
Topic: Digestion
Materials:
Plasmid vector PSB1C3 10 μl
Promoter (pncrA) 10 μl
Repressor protein (NcrB) 10 μl
restriction enzyme (Ecor1) 1 μl
restriction enzyme (Pst1) 1 μl
10 × Buffer O 2μ
Agarose 0.6 g
1×TAE 60ml
1/10000 Bio Teke Dye 6 μl
2000bp maker 8μl
10 × Buffer 3 μl
DNA purification recovery kit (TIANGEN company)
Procedure:
Digestion System:1 μl of Ecor1, 1 μl of Pst1, 10 μl of digested DNA, 10 × Buffer O 2 μ, 6 μl of distilled water, total 20μl.
Configure the mixture according to the system
37 ° C for 2 hours
65 ℃ fire for 20 minutes
Purification of the digestion product using a DNA purification recovery kit and running gel.
Results and analysis:
Digestion effect is not too good and the band is not bright enough.
Probably because the concentration of the target fragment we added was insufficient.
Fig7. Digestion of Pncra and Ncrb
None. Results are expected after transformation and PCR.
7.29-8.4
Topic:Ligation
Materials:
Digested PSB1C3 2μl
Digested NcrB 5μl
Digested pncrA 8μl
T4 DNA ligase 1μl
10xT4 Buffer 2μl
DD H2O 19μl
Procedure:
Connection system: The total volume is 20μl
Linear plasmid vector: 1μl，about 30 ng (measured plasmid backbone concentration of 29.2 ng /μl)
Inserted DNA:plasmid backbone = 1: 1-5: 1 ，and we select ratio of 3: 1
Digested NcrB: 5μl (measured at a concentration of 16.7 ng /μl)
Or digested pncrA: 8μl (measured at a concentration of 11 ng /μl)
10xT4 Buffer: 2μl
T4 DNA ligase: 1μl (Thermo Corporation, No. EL0014, enzyme activity 5 weiss U /μl)
added DD H2O to 20 μl
Configure the mixture according to the system above
22 °C for 40 minutes
65 °C inactivated for 20 minutes
Results and analysis:
None. Results are expected after transformation and PCR.
Topic: Transformation
Materials:
Recombinant plasmid of ncrB DNA 2 μl
Recombinant plasmid of pncrA DNA 2 μl
DH5α bacterium 200 μl
SOC 800 μl
Chloramphenicol 200 μl
Agar Petri dish 8
Procedure:
1. Take a commercial competent cells (total 100μl), two self-made competent cells (each 80μl), put frozen on ice 15min
2. Prepare four sterilized 2mlEP tubes, put on ice to precool (This should be carried out at the same time with the first step )
3. Draw 50 μl of competent cell fluid added to the four precooled EP tube (two commercial, two self-made)
4. Add 1μl recombinant plasmid to each of the commercial competent cells and self-made competent cells. Do the same to the pncrA DNA.
5. Close the EP tube, and then put it in an ice bath for 30min
6. 42°C heat shock for 1min, put it in an ice bath for 5min
7. Add 200μl of SOC medium to each EP tube
8. 37 °C shaker culture 3h
9. Take two samples, one 50 μl and one 150 μl, of each EP tube, place onto the plate and label them.
10. place in incubator at 37C for 12 to 16 hours.
Results and analysis:
Fig 7.Transformation of pncrA
Fig8.Transformation of NcrB
The recombinant plasmid with pncrA didn’t transform into E. coli, the recombinant plasmid with ncrB transformed into E. coli successfully. The DNA gel electrophoresis results showed digestion succeeded, thus there was no conoly due to the failure of ligation and transformation.
Topic: PCR of pncrA
Materials:
2 × taq enzyme 75 μl
pncrA forward primer 1.5 l
pncrA reverse primer 1.5 μl
DD H2O 72μl
Agarose 0.3g
1 × TAE 30ml
1/10000 Bio Teke Dye 3 μl
2000bp maker 4μl
10 × Buffer 6 μl
bacteria
Procedure:
PCR MIX system: the total volume is 25μl. 2×taq enzyme 25μl ( from Engine company), 0.5μl forward and 0.5μl reverse primer, DDH2O 24μl, the total is 50μl. Then divide them into two tubes. Repeat the operations for other two times, and there are 6 tubes totally.
A single colony was randomly selected at room temperature, and was picked with a sterile toothpick or a tip, and a copy was made on a solid plate and added to 500μl of liquid LB medium. The colonies were added to the PCR MIX system in the same manner. Do a colony PCR to verify the part size.
Results and analysis:
Fig9. PCR of Pncra
There was a successful connection of pncrA DNA bands, which may have a small amount of specific amplification. the recombinant plasmid with ncrB transformed into E. coli successfully, the problem may be either ligation or Transformation, but ligation is more likely.
8.4-8.11
Topic:Ligation
Materials:
Digested PSB1C3 4μl
Digested NcrB 6μl
Digested pncrA 6μl
T4 DNA ligase 2μl
10xT4 Buffer 2μl
Procedure:
Connection system: The total volume is 10μl.
Linear plasmid vector: 2μl
Interested DNA: plasmid backbone = 1: 1-5: 1 ，and we select ratio of 3: 1
Digested Ncrb: 6μl
Or digested Pncra: 6μl
10xT4 Buffer: 1μl
T4 DNA ligase: 1μl
added DD H2O to 20 μl
Configure the mixture according to the system
22 °C for 4 hours
65 ℃ inactivated for 30 minutes
Results and analysis:
None. Results are expected after transformation and PCR.
Topic:Transformation
Materials:
ligated plasmid of ncrB DNA 2 μl
ligated plasmid of pncrA DNA 2 μl
DH5α bacterium 100 μl
Chloramphenicol 200 μl
LB medium 1 mL
LB solid medium 50 mL
Chloramphenicol 50 μl
Agar Petri dish 2
Procedure:
1. Take two self-made competent cells (each 80μl) and put on ice to freeze - 15min
2. Prepare two sterilized 2ml EP tubes, put on ice to precool (This should be carried out at the same time as the first step )
3. Draw 50 μl of competent cell fluid and add to the two precooled EP tubes
4. Add 1μl recombinant plasmid to each of the commercial competent cells and self-made competent cells. Do the same to the pncrA DNA.
5. Close the EP tube, and then put it in an ice bath for 30min
6. 42°C heat shock for 90s, put it in an ice bath for 5min
7. Add 200 μl of SOC medium to each EP tube
8. 37 ℃ shaker culture 3h
9. Take 50 μl and 150 μl of each EP tube onto the plate and label them
10. 37 ° C incubating for 12 to 16 hours
Results and analysis:
Fig 10. Transformation of pncrA and ncrB
DH5α were successfully transformed and the colonies were grown on the medium.
Topic:PCR of pncrA and ncrB
Materials:
2xtaq enzyme 125μl
pncrA forward primer 2.5μl
pncrA reverse primer 2.5μl
ncrB forward primer 2.5μl
ncrB reverse primer 2.5μl
DD H2O 120μl
bacteria
Agarose 0.6g
1 × TAE 60ml
1/10000 Bio Teke Dye 6 μl
2000bp maker4μl
10 × Buffer 12 μl
Procedure:
PCR MIX system: the total volume is 25μl.
2×taq enzyme 25μl (from Engine company),
0.5μl forward and reverse primer,
DD H2O 24μl, the total is 50μl.
Then divide them into two tubes. Repeat the operations for other 5 times, and there are 10 tubes totally.
A single colony was randomly selected at room temperature, and was picked with a sterile toothpick or a tip, and a copy was made on a solid plate and added to 500μl of liquid LB medium. The colonies were added to the PCR MIX system in the same manner. Do a colony PCR to verify the part size.
Results and analysis:
Fig11. PCR of pncrA and ncrB
We improve the ligation system, extend the ligation time, optimize the transformation step. We try to make ncrB DNA, pncrA and plasmid vector connected and its successful transformation.
Topic:sequencing
Procedure:
We will send the samples to company for further sequencing.
Results and analysis:
Fig12.Comparison of ncrB
Fig13.Comparison of pncrA
After sequencing, we confirmed that the DNA is indeed what we need.
8.12-8.23
Topic: Digestion，Ligation，Ligation
Procedure:
The method of digestion and ligation is the same as above.
Transformation: The system and method are the same as before, except that the colonies are not required to be coated with a solid culture medium and can be directly cultured on a liquid medium overnight to extract the plasmid. The transformed plasmids are E1010, B0010, B0012, B0032, B0034, J23102.
Results and analysis:
B0010 failed to convert and there were no Colonies, but other plasmids successfully transformed.
Topic:3A assembly
Materials:
Linear plasmid vector (PSB1T3) 10μl
J23102 10μl
B0032 10μl
B0034 10μl
Digested of pncrA 5μl
T4 DNA ligase 4μl
10xT4 Buffer 3μl
restriction enzyme (EcoRI) 4μl
restriction enzyme (Pst1) 4μl
Restriction enzyme (XbaI) 2μl
restriction enzyme (SpeI) 2μl
10 × Buffer O 12μl
10 × Buffer T 6μl
DD H2O 18μl
Procedure:
The method of digestion, ligation and transformation is the same as above. And then use the mention the plasmid kit.
Fig14. Principle of 3A assembly
The clips are joined in this order:
J23102-B0032-ncrB-B0010-B0012-pncrA-B0034-E1010-B0012-B0034.
We first use the 3A method toconnect thesefragments:
J23102-B0032-ncrB; B0010-B0012-Pncra-B0034; E1010-B0012-B0034.
The "three large fragments" were then ligated using Gibson assembly technology (seamless splicing), and finally transformed into the PSB1C3 plasmid.
Results and analysis:
Because of limited time, we regret that we have not been able to complete all the experiments. but we have successfully confirmed the J23102-B0032 connection to the feasibility of our idea.
